HT RNA Pico Sensitivity LabChip Kit LabChip GX/GXII User Guide

Transcription

1 Contents SPECIFICATIONS... 2 SAMPLE CONDITIONS... 2 KIT CONTENTS... 3 SAFETY WARNINGS AND PRECAUTIONS... 4 PREPARATION PROCEDURES... 5 ADDITIONAL ITEMS REQUIRED... 5 PREPARING LADDER ALIQUOTS... 5 PREPARING THE RNA CHIP... 6 PREPARING THE RNA SAMPLES, LADDER AND BUFFER TUBE... 8 RUNNING THE RNA PICO SENSITIVITY ASSAY... 9 STORING THE RNA CHIP RESULTS HT RNA PICO SENSITIVITY LADDER RESULT HT RNA PICO EUKARYOTE SAMPLE RESULT HT RNA PICO MRNA SAMPLE RESULT TROUBLESHOOTING LABCHIP KIT ESSENTIAL PRACTICES GENERAL REAGENTS CHIPS SAMPLES CHIP WELL ASPIRATION USING A VACUUM CUSTOMER TECHNICAL SUPPORT Caliper Life Sciences (a PerkinElmer company) Page 1 of 22 Revised August 2012

2 Specifications 1 Sensitivity Linear Concentration Range Quantitation Reproducibility Quantitation Accuracy Size Range 250 pg/µl Total RNA 500 pg/ul mrna pg/µl Total RNA pg/ul mrna 20% CV ± 30% (for ladder as sample) nucleotides RNA Sample Volume 2 µl Carry-Over Run Time Compatible Plate Types < 0.5% following 5 ng/µl sample 80 seconds per sample (about 2.5 hours for 96 samples) 384-well Number of Samples per Chip Prep Up to 96 Chip Lifetime samples per chip Samples per Reagent Kit up to 480 Sample Conditions Additives Caliper recommends that BSA and detergents exceeding 0.05 mg/ml and 0.01% (v/v) respectively in concentration not be used. Higher concentrations can result in chip failure. In addition, non-aqueous solvents are not compatible with LabChip protocols. Particulates All sample plates should be spun down prior to analysis. All buffers should be filtered with a 0.22 µm cellulose acetate filter. Salt Concentration Total salt concentration must not exceed 10 mm Tris. Higher salt concentrations and different ions may alter performance and reduce assay sensitivity. 1 All specifications pertaining to Total RNA and mrna were determined using RNA diluted in water. 2 Expected chip lifetime is based on use under normal laboratory conditions and adherence to Caliper preparation protocols, sample guidelines and storage conditions. Individual results may vary. Caliper Life Sciences (a PerkinElmer company) Page 2 of 22 Revised August 2012

3 Kit Contents HT RNA Pico Sensitivity Reagent Kit Part Number Kit contains enough reagents for 9 Medium-throughput (MT) or 5 High-throughput (HT) chip preparations. Up to 48 samples can be tested with a MT chip prep. Up to 96 samples can be tested with a HT chip prep. Reagent Vial Quantity RNA Dye Concentrate Blue 1 vial, 0.5 ml Chip Storage Buffer White 4 vials, 1.8 ml each RNA Pico Gel Matrix Red 2 vials, 1.5 ml each RNA Pico Ladder 1 Yellow 1 vial, 0.04 ml (packaged separately, Part Number CLS760652) RNA Pico Marker Green 1 vial, 0.8 ml 10X RNA Sample Buffer Concentrate Purple 3 vials, 1.8 ml each Consumable Items Supplier Number Quantity Spin Filters Costar, Cat. # Centrifuge Tubes Eppendorf, Cat # , 2.0 ml Ladder Tubes Genemate, Cat. # C , 0.2 ml Detection Window Cleaning Cloth VWR, Cat. # Swab ITW Texwipe, Cat. # TX758B 3 Buffer Tubes E&K Scientific, Cat. # NC 10, 0.75 ml HT RNA Chip Part Number Item Quantity RNA Chip 1 1 Additional RNA Pico Ladder can be ordered separately using Part Number CLS Caliper Life Sciences (a PerkinElmer company) Page 3 of 22 Revised August 2012

4 Safety Warnings and Precautions! WARNING! For Research Use Only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. CAUTION We recommend that this product and components be handled only by those who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. As all chemicals should be considered as potentially hazardous, it is advisable when handling chemical reagents to wear suitable protective clothing, such as laboratory overalls, safety glasses, and gloves. Care should be taken to avoid contact with skin or eyes. In case of contact with skin or eyes, wash immediately with water.! WARNING! Dye Concentrate contains DMSO. S24/25: Avoid contact with skin and eyes. Storage: When not in use, store chips and reagents refrigerated at 4 C. Do not leave chips and reagents unrefrigerated overnight. Caliper Life Sciences (a PerkinElmer company) Page 4 of 22 Revised August 2012

5 Preparation Procedures HT RNA Pico Sensitivity LabChip Kit Additional Items Required MilliQ water: Molecular biology grade or better, 0.22-micron filtered 70%-isopropanol solution in DI water DEPC treated water (nuclease-free) Note: Allow the chip and reagents to equilibrate to room temperature for about 20 minutes before use. The Dye Concentrate must be completely thawed and vortexed before use. The RNA Pico Ladder should be kept on ice. Avoid multiple freeze-thaws. Preparing Ladder Aliquots 1. Spin down RNA Pico Ladder and heat denature at 70 C for 2 minutes. Afterward immediately snap cool on ice for 5 minutes. 2. Prepare 4 µl aliquots in nuclease-free tubes. Store aliquots at -70 C. 3. Upon use of frozen aliquots do not heat denature again. If needed additional ladder can be ordered (Part Number CLS760652). Caliper Life Sciences (a PerkinElmer company) Page 5 of 22 Revised August 2012

6 Preparing the RNA Chip Medium-Throughput Chip Preparation (For up to 48 samples) 1. Vortex thawed RNA Pico Gel Matrix (red cap ) and RNA Dye Concentrate (blue cap ). 2. Prepare Gel-Dye by transfering 255 µl RNA Pico Gel Matrix (red cap ) to a nuclease-free tube using a Reverse Pipetting Technique (for more details see LabChip Kit Essential Practices section). 3. Add 45 µl RNA Dye Concentrate (blue cap ) to the gel matrix. Vortex until solution is well mixed and transfer to a spin filter. 4. Centrifuge at 9200 rcf for 10 minutes at room temperature. Make sure that all of the gel has passed through the filter and then discard the filter. (Note: Gel-dye can be stored in the dark at 2-8 C for 4 days.) 5. Use a pipette tip attached to a vacuum line to thoroughly aspirate all fluid from the chip wells (as shown in Figure 1). For more details on how to setup a vacuum line see page Rinse and aspirate each active well (1, 3, 4, 7, 8 and 10) twice with nuclease-free water. Do not allow active wells to remain dry. 7. If any water spilled onto on the top and bottom chip surfaces during rinsing aspirate using vacuum line. DO NOT run the tip over the central region of the detection window. 8. Add prepared Gel-Dye to chip wells 3, 7, 8 and 10 (as shown in Figure 2) using a Reverse Pipetting Technique. 9. Add 50 µl RNA Pico Marker (green cap ) to chip well 4 (as shown in Figure 2). (Note: The marker well may need to be replenished if the chip is in idle mode on the instrument for an extended period of time.) 10. Ensure chip well 1 (or waste well) is empty before placing the chip on the LabChip GX or GXII. Figure 1. Using a vacuum to aspirate the chip wells is more effective than using a pipette. Waste Well Figure 2. Medium-Throughput Chip Preparation Caliper Life Sciences (a PerkinElmer company) Page 6 of 22 Revised August 2012

7 High-Throughput Chip Preparation (For up to 96 samples) 1. Vortex thawed RNA Pico Gel Matrix (red cap ) and RNA Dye Concentrate (blue cap ). 2. Prepare Gel-Dye by transfering 425 µl RNA Pico Gel Matrix (red cap ) to a nuclease-free tube using a Reverse Pipetting Technique (for more details see LabChip Kit Essential Practices section). 3. Add 75 µl RNA Dye Concentrate (blue cap ) to the gel matrix. Vortex until solution is well mixed and transfer to a spin filter. 4. Centrifuge at 9200 rcf for 10 minutes at room temperature. Make sure that all of the gel has passed through the filter and then discard the filter. (Note: Gel-dye can be stored in the dark at 2-8 C for 4 days.) 5. Use a pipette tip attached to a vacuum line to thoroughly aspirate all fluid from the chip wells (as shown in Figure 1). For more details on how to setup a vacuum line see page Rinse and aspirate each active well (1, 3, 4, 7, 8 and 10) twice with nuclease-free water. Do not allow active wells to remain dry. 7. If any water spilled onto on the top and bottom chip surfaces during rinsing aspirate using vacuum line. DO NOT run the tip over the central region of the detection window. 8. Add prepared Gel-Dye to chip wells 3, 7, 8 and 10 (as shown in Figure 3) using a Reverse Pipetting Technique. 9. Add 75 µl RNA Pico Marker (green cap ) to chip well 4 (as shown in Figure 3). (Note: The marker well may need to be replenished if the chip is in idle mode on the instrument for an extended period of time.) 10. Ensure chip well 1 (or waste well) is empty before placing the chip on the LabChip GX or GXII. Waste Well Figure 3. High-Throughput Chip Preparation Caliper Life Sciences (a PerkinElmer company) Page 7 of 22 Revised August 2012

8 Preparing the RNA Samples, Ladder and Buffer Tube Note: Due to sample evaporation test up to 48 samples only per run. For example if analyzing 96 samples, test samples in a total of 2 runs. Total salt concentration of samples must not exceed 10 mm Tris. Higher salt concentrations and different ions may alter performance and reduce assay sensitivity. Pipette 8µL Sample Buffer into a 384-well plate. Add 2µL sample and mix well. Tightly seal and spin down plate. Denature by heating the samples at 70 C for 2 min. Immediately after snap cool on ice for 5 min. Spin down plate. Thaw 4µL aliquot of RNA Ladder. Add 116 µl Sample Buffer to RNA Ladder. Mix thoroughly and transfer solution to the provided Ladder Tube. Ladder Tube Pipette 750 µl Sample Buffer in the provided Buffer Tube. LabChip GX/GXII Buffer Tube Figure 3. Sample, Ladder Tube and Buffer Tube Preparation 1. Prepare Sample Buffer by adding 200 µl RNA Sample Buffer Concentrate to 1800 µl DEPC treated or nuclease-free water. (Note: The RNA Sample Buffer Concentrate is a 10X solution. Sample Buffer is stable after dilution, but to avoid RNase contamination sample buffer should be prepared fresh.) 2. In a 384-well plate pipette 8 µl Sample Buffer to an equivalent number of wells as samples being tested (but to no more than 48 wells). Add 2 µl sample to each well and mix by pipetting up and down. Tightly seal and spin down plate. 3. Denature samples by heating at 70 C for 2 minutes. Immediately after snap cool on ice for 5 minutes. Spin down plate. (Note: For sample heat denature, if a 384-well thermocycler or heat block is not available, sample plate can be heated by placing plate on top of one heat block, and then placing another heat block on top of the plate.) 4. Prepare RNA Ladder by thawing a 4 µl aliquot. Add 116 µl Sample Buffer to RNA Ladder aliquot. Mix thoroughly and transfer solution to the provided Ladder Tube. 5. Pipette 750 µl Sample Buffer into Buffer Tube. Caliper Life Sciences (a PerkinElmer company) Page 8 of 22 Revised August 2012

9 Inserting a Chip into the LabChip GX Instrument 1. Place the sample plate, Ladder Tube and Buffer Tube onto the plate holder of the LabChip GX. 2. Remove the chip from the chip storage container and inspect the chip window. If needed clean BOTH sides of the chip window with the Calipersupplied cleanroom cloth dampened with a 70%- isopropanol solution in DI water. 3. Eject the chip cartridge by pressing the CHIP button on the instrument front panel. HT RNA Pico Sensitivity LabChip Kit 4. Release the cartridge latch, insert the chip into the LabChip GX instrument, refasten the latch and push the cartridge into the instrument. 5. Press the EJECT button on the instrument front panel to retract the sample plate and send the Buffer Tube to the sipper. Ladder Tube Sample Well A1 Buffer Tube Note: If performing multiple runs in a day, in between chip preparations chip should be washed using the instrument and Chip Storage buffer as described in Storing the RNA Chip section. Be sure to periodically clean the O-rings on the top plate of the chip interface on the LabChip GX instrument. Use the provided lint free swab dampened with DI water to clean the O-rings, using a circular motion. Allow the O-rings to dry before inserting a chip. Running the RNA Pico Sensitivity Assay 1. Start the LabChip GX software. 2. On the main screen, click on the Run button in the upper left corner of the LabChip GX Software. 3. The Start Run dialog box will pop up with tabs listed as Output, Run and Advanced. 4. In the Run Tab, select or enter the appropriate assay type, operator name, plate name, well pattern and barcode option. For HT RNA Pico Sensitivity the available assay types are: HT RNA Pico Sensitivity: For analysis of Eukaryotic Total RNA. HT RNA Pico mrna: For analysis of mrna. 5. In the Output Tab select the destination of the file, the filename convention and any additional data to autoexport. 6. In the Advanced Tab, select the number of times each well is sampled, the inclusion of any sample names and any expected peaks. 7. Click Start to begin the run. Caliper Life Sciences (a PerkinElmer company) Page 9 of 22 Revised August 2012

10 Storing the RNA Chip After use the chip must be cleaned and stored in the chip container. The procedure below can be conducted the following day when running overnight. 1. Remove the reagents from each well of the chip using vacuum. 2. Rinse and aspirate each active well (1, 3, 4, 7, 8, and 10) twice using molecular biology grade water. 3. Add 100 µl of HT RNA Chip Storage Buffer (white cap ) to the active wells. 4. Place the chip in the LabChip GX instrument and click the Wash button in the left corner of the LabChip GX Software. Ensure a Buffer Tube with 750 µl buffer or molecular biology grade water is in the buffer slot. 5. Remove the chip from the instrument and place in container. 6. Add an additional 50 µl of Storage Buffer to wells 1 and Cover the wells with Parafilm to prevent buffer evaporation and store at 4 C. Storage of a chip with dry wells may cause it to become clogged. Chip Cartridge Cleaning 1. Daily A) Inspect the inside of the chip cartridge and O-rings for debris. B) Use the provided lint free swab dampened with DI water to clean the O-rings using a circular motion. If the O-rings stick to the chip or a pressure leak is detected, perform the more extensive monthly cleaning procedure. 2. Monthly A) To reduce pressure leaks at the chip interface, clean the O-rings frequently. Remove the O-rings from the top plate of the chip interface on the LabChip GX instrument. Soak O-rings in DI water for a few minutes. Clean the O-ring faces by rubbing between two fingers. B) To reduce the occurrence of current leaks, clean the chip interface frequently. Clean the top plate of the chip interface using the provided lint free swab dampened with DI water. C) Allow the O-rings and chip interface to air dry. Reinsert the O-rings into the chip cartridge. Caliper Life Sciences (a PerkinElmer company) Page 10 of 22 Revised August 2012

11 Results HT RNA Pico Sensitivity Ladder Result The electropherogram of a typical RNA Pico Sensitivity ladder is shown below. Following the lower marker, ladder fragments in order of increasing migration time correspond to 200, 500, 1000, 1500, 2000, 3000, 4000 and 6000 nt. Lower Marker HT RNA Pico Eukaryote Sample Result The electropherogram for a typical total RNA sample is shown below. Your results may vary depending on the type of total RNA. Lower Marker Caliper Life Sciences (a PerkinElmer company) Page 11 of 22 Revised August 2012

12 HT RNA Pico mrna Sample Result HT RNA Pico Sensitivity LabChip Kit The electropherogram for a typical mrna sample is shown below. Your results may vary depending on the type and concentration of mrna. Lower Marker Caliper Life Sciences (a PerkinElmer company) Page 12 of 22 Revised August 2012

13 Troubleshooting 1. Symptom: No ladder or sample peaks but Lower Marker peak detected. The lower marker peak height will most likely be greater than normal height. Possible causes: A) Air bubble in sipper introduced during chip priming. B) A combination of problems described in #2 and #3 (see below). What to do: A) Reprime the chip. See the section entitled LabChip Kit Essential Practices Chips for instructions on how to reprime the chip. Lower Marker 2. Symptom: Ladder detected but no sample peaks Possible causes: A) The sipper is not reaching the sample due to low sample volume in the well plate. B) If the missing sample peaks occurred only in a few wells of the plate, check those wells for air bubbles. C) The sipper is not reaching the sample due to an incorrect capillary height setting. D) If the plate has been uncovered for some time, sample evaporation might have occurred. E) Debris from the sample or sample preparation is clogging the sipper. Caliper Life Sciences (a PerkinElmer company) Page 13 of 22 Revised August 2012

14 What to do: A) Add more sample to the well or dilute sample with water or 1X TE buffer. Recommended sample volumes are 15 µl for a 384-well plate or 40 µl for a 96-well plate, when a full plate is run. B) Manually insert a larger volume pipette tip (~100 µl) into the sample well and dislodge the bubble. Rerun these sample wells. C) Re-teach the robot positioning as described in LabChip GX User s Manual. D) Check the sample wells, especially around the edge of the plate where evaporation is fastest, and replenish low volume wells. Add or dilute samples. E) If you suspect there may be debris in your samples, spin the sample plate down in a centrifuge. Unclog the sipper by repriming the chip. See the section entitled LabChip Kit Essential Practices Chips for instructions on how to reprime the chip. 3. Symptom: No ladder peaks but sample peaks and marker peaks are present. Possible causes: A) Low or no ladder volume in Ladder Tube. What to do: A) Add more ladder to the Ladder Tube and restart the run. Recommended minimum ladder volume is 100 µl. 4. Symptom: No marker peaks but sample peaks are present. Possible causes: A) No marker added to chip well 4. B) If there is marker solution in well 4, the problem may be due to a marker channel clog. What to do: A) This may be due to not filling marker well or chip remaining idle on instrument for extended period of time. Add or replenish the marker solution in the chip using the following procedure: Press the CHIP button on the front instrument panel to eject the chip cartridge. Open the chip cartridge and return the chip to the chip container ensuring the sipper is immersed in fluid. Thoroughly aspirate all fluid from chip well 4 using a vacuum line. Ensure that chip well 4 is rinsed and completely aspirated twice with molecular biology grade water. Add 50 or 75 µl HT RNA Pico Marker (green cap) to chip well 4. Please note that the marker well may need to be replenished if the chip is in idle mode on the instrument for an extended period of time. Place the chip in the LabChip GX instrument. Caliper Life Sciences (a PerkinElmer company) Page 14 of 22 Revised August 2012

15 Reinsert the cartridge by engaging the latch and pushing the cartridge back into the instrument. Press the Run button on the main screen of the LabChip GX software. B) Perform a marker channel unclogging procedure by repriming the chip. See the section entitled LabChip Kit Essential Practices Chips for instructions on how to reprime the chip. 5. Symptom: Peaks migrating much faster than expected. NOTE: Some migration time variance between chips or within a plate is considered normal chip performance. All chips are QC tested at Caliper Life Sciences prior to shipment, and a normal migration time window for the lower marker is listed below: HT RNA Pico Sensitivity assay: Lower Marker migration time window is 28 to 33 seconds. Possible causes: A) Incorrect Gel-Dye concentration. Migration time is sensitive to dye concentration and peaks will migrate too fast or too slow if the dye concentration in the gel is too low or too high, respectively. What to do: A) Prepare a fresh Gel-Dye solution. Wash and reprime the chip with the new Gel-Dye mixture. See the section entitled LabChip Kit Essential Practices Chips for instructions on how to wash and reprime the chip. B) If fast migration is observed repeatedly on a new chip then return the chip to Caliper Life Sciences along with data file for replacement. Caliper Life Sciences (a PerkinElmer company) Page 15 of 22 Revised August 2012

16 6. Symptom: Peaks migrating much later than expected. The electropherogram of a RNA Pico ladder is shown below where the migration time is much longer than expected. Lower Marker Possible causes: A) Particulates from the samples may be clogging the separation channel. B) Excess dye within the separation channel. C) Gel-Dye was not primed properly into the chip. What to do: A) Minimize the loading of particulates in the sample by performing a centrifuge spin of the sample plate (e.g rcf for 5 min) and/or selecting a plate type with a higher sip height in the Start Run dialog box before starting a new run. The debris maybe flushed out of the chip by washing and re-priming the chip. See the section entitled LabChip Kit Essential Practices Chips for instructions on how to wash and reprime the chip. B) Prepare a fresh Gel-Dye solution. Wash and reprime the chip with the new Gel-Dye mixture. See the section entitled LabChip Kit Essential Practices Chips for instructions on how to wash and reprime the chip. C) Check the O-rings on the top surface of the chip interface and clean if necessary. Caliper Life Sciences (a PerkinElmer company) Page 16 of 22 Revised August 2012

17 7. Symptom: Unexpected sharp peaks. HT RNA Pico Sensitivity LabChip Kit The electropherogram of a mrna sample is shown below with an unexpected sharp peak caused by dust or other particulates. Lower Marker Peak caused by particulates Possible causes: A) Dust or other particulates introduced through sample or reagents. What to do: A) Replace molecular biology grade water used for chip preparation. Replace buffer used for sample and reagent preparation. Use a 0.22-micron filter for all water and buffers used for chip, sample and reagent preparation. Caliper Life Sciences (a PerkinElmer company) Page 17 of 22 Revised August 2012

18 LabChip Kit Essential Practices HT RNA Pico Sensitivity LabChip Kit To ensure proper assay performance please follow the important handling practices described below. Failure to observe these guidelines may void the LabChip Kit product warranty. 1 NOTE: It is important to keep particulates out of the chip wells, channels and capillary. Many of the following guidelines are designed to keep the chips particulate free. For assay and instrument troubleshooting, refer to the LabChip GX Software Help file or call Caliper Technical Support at LABCHIP. General Allow the chip, sample plate and all reagents to equilibrate to room temperature before use (approximately 20 to 30 minutes). Clean the O-rings in the chip interface weekly and the electrodes daily. Refer to the Instrument Users Guide Maintenance and Service section for procedures. Avoid use of powdered gloves. Use only non-powdered gloves when handling chips, reagents, sample plates, and when cleaning the instrument electrodes and electrode block. Calibrate laboratory pipettes regularly to ensure proper reagent dispensing. Only the Caliper-supplied clean room cloth can be used on the chip to clean the detection window. Use of other, non-approved wipes may leave fluorescent debris, which can cause erratic focusing. Water used for chip preparation procedures must be 0.22-micron filtered deionized, molecular biology grade. Use of the Reverse Pipetting Technique (described below) will help avoid introducing bubbles into the chip when pipetting gel or other viscous solutions. Reverse Pipetting Technique Step 1. Depress the pipette plunger to the second stop. Step 2. Aspirate the selected volume plus an excess amount from the tube. Step 3. Dispense the selected volume into the corner of the well by depressing plunger to the first stop. Step 4. Withdraw the pipette from the well. Reagents Store all reagents at 4 C when not in use. The LabChip dye contains DMSO and should be thawed completely before use. It is recommended that you prepare aliquots to reduce the time required for thawing. Gently vortex all kit reagents. 1 Caliper Life Sciences warrants that the LabChip Kit meets specification at the time of shipment, and is free from defects in material and workmanship. LabChip Kits are warranted for 90 days from the date of shipment. All claims under this warranty must be made within thirty days of the discovery of the defect. Caliper Life Sciences (a PerkinElmer company) Page 18 of 22 Revised August 2012

19 Dispense reagents into chip wells slowly without introducing air bubbles. Insert the pipette tip vertically and to the bottom of the chip well. Protect the dye, Gel-Dye mixture and Marker solution from light. Store in the dark at 4 C when not in use. Chips Repriming Chips: Press the CHIP button on the front instrument panel to eject the chip cartridge. Reinsert the cartridge by pushing the cartridge back into the instrument. Press the Run button on the main screen of the LabChip GX software. Washing and Repriming Chips: Press the CHIP button on the front instrument panel to eject the chip cartridge. Open the chip cartridge and return the chip to the chip container ensuring the sipper is immersed in fluid. Thoroughly aspirate all fluid from the chip wells using a vacuum line. Ensure that each active well (1, 3, 4, 7, 8 and 10) is rinsed and completely aspirated twice with molecular biology grade water. Do not allow active wells to remain dry. Add 100 µl of Chip Storage Buffer to each active well (1, 3, 4, 7, 8 and 10). Place the chip in the LabChip GX instrument. Reinsert the cartridge by engaging the latch and pushing the cartridge back into the instrument. Ensure a Buffer Tube with 750 µl buffer or molecular biology grade water is in the buffer slot. Press the Wash button on the main screen of the LabChip GX software. After completion of the wash cycle, Press the CHIP button on the front instrument panel to eject the chip cartridge. Open the chip cartridge and return the chip to the chip container ensuring the sipper is immersed in fluid. Thoroughly aspirate all fluid from the chip wells using a vacuum line. Ensure that each active well (1, 3, 4, 7, 8 and 10) is rinsed and completely aspirated twice with molecular biology grade water. Do not allow active wells to remain dry. Re-prep chip according to instructions in Preparing the RNA Chip section. Place the chip in the LabChip GX instrument. Reinsert the cartridge by engaging the latch and pushing the cartridge back into the instrument. Press the Run button on the main screen of the LabChip GX software. Other Considerations: Chips should be stored refrigerated prior to first use. Caliper Life Sciences (a PerkinElmer company) Page 19 of 22 Revised August 2012

20 Cover the active wells on the chip with Parafilm and store at 4 C. If using the chip again within 24 hours it may be left at room temperature. Do not allow the liquid in the chip container to freeze, as this may lead to poor chip performance. Do not submerge the chip in any solution. The entire chip surface must be thoroughly dry before use. The sipper must be kept immersed in fluid at all times and should not be exposed to an open environment for long periods of time. Use care in chip handling to prevent sipper damage. Damage to the sipper can result in inconsistent sampling. Avoid exposing the chips to dust by keeping them in a closed environment such as in the chip container or in the instrument before and after chip preparation. Chips can be prepared and left idle on the instrument for up to 8 hours. This workflow allows analysis of samples as needed throughout the day without having to re-prep the chip as long as the maximum number of samples per chip prep is not exceeded. Samples Prepared sample plates should be free of gas bubbles and particulate debris, both of which may inhibit sipper flow. Sample plates containing gas bubbles and/or particulate debris should be spun down (for example, 3000 rcf for 5 min at RT) prior to analysis. Caliper Life Sciences (a PerkinElmer company) Page 20 of 22 Revised August 2012

21 Chip Well Aspiration Using a Vacuum HT RNA Pico Sensitivity LabChip Kit Aspirating with a pipette can leave used reagents in the chip wells. For this reason, Caliper recommends vacuuming the wells instead. This can be accomplished by attaching a permanent pipette tip to a house vacuum line with trap (Figures 1a and 1b). To avoid contamination, use a new pipette tip over the permanent tip for each chip aspirated (Figure 2). Figure 1a Figure 1b Figure 2 Caliper Life Sciences (a PerkinElmer company) Page 21 of 22 Revised August 2012

22 Customer Technical Support Caliper Life Sciences Phone: Fax: HT RNA Pico Sensitivity LabChip Kit For additional assay and instrument troubleshooting, refer to the LabChip GX Software Help file. Call Caliper Technical Support at LABCHIP. The chip and reagents supplied with this kit are sold with limited rights of use. The chip may only be used with the specific quantity of reagents supplied with this kit. The purchaser has no right or license to refurbish, reuse, remanufacture, or otherwise use the chip with any other reagents than those specifically supplied in this kit. For more information on the terms and conditions of use of these chips and reagents, please read your LabChip GX User Guide. Caliper, the Caliper logo, LabChip, and the LabChip logo are registered trademarks of Caliper Life Sciences, a PerkinElmer company. The reagent kits contain materials manufactured for Caliper by Molecular Probes, Inc., and are provided under a license from Molecular Probes, Inc., for only use in Research, Human Diagnostics, Biohazard Detection, Environmental Testing, Food Testing, Quality Control, and Pathogen Testing. Copyright Caliper Life Sciences Doc CLS Caliper Life Sciences (a PerkinElmer company) Page 22 of 22 Revised August 2012